ABSTRACT To sustain hematopoiesis, hematopoietic stem cells (HSCs) must persist throughout life, constantly regenerating hematopoietic cells lost to normal turnover, bleeding, and disease. Much has been learned over the past ten years regarding the mechanisms that regulate HSC maintenance. This work has demonstrated that several aspects of cellular physiology are regulated differently in HSCs as compared to other hematopoietic cells. This raises the fundamental question of whether all aspects of cellular physiology are regulated differently in stem cells as compared to restricted progenitors, or whether certain aspects of cellular physiology are house-keeping functions that are regulated similarly in stem cells and restricted progenitors. Unfortunately, many aspects of cellular physiology are technically difficult to study with existing techniques in small numbers of stem cells and therefore have not yet been addressed, leaving large areas of biology unexplored. One such aspect of cellular physiology is the regulation of protein synthesis. There are almost no data on the regulation of translation in any somatic stem cell population, partly because assays have not yet been developed to study translation in small numbers of cells in vivo. We have recently developed an assay that makes it possible to study the rate at which polypeptides are synthesized by individual cells in vivo. Using this assay we have determined that HSCs have significantly lower rates of protein synthesis than other hematopoietic cells even when we control for differences in cell cycle distribution. Our preliminary functional data suggest that HSC maintenance depends upon highly regulated rates of protein synthesis. This discovery may explain previously observed defects in HSC self-renewal that were not understood at a mechanistic level. For example, we have demonstrated previously that deletion of the PTEN tumor suppressor in adult hematopoietic cells increases PI3-kinase pathway signaling in HSCs, leading to leukemogenesis and HSC depletion. Although the depletion of PTEN deficient HSCs is known to depend upon a tumor suppressor response induced by mTORC1 and mTORC2 signaling, it is unknown how elevated mTOR signaling increases tumor suppressor expression. In this application we propose to extend our preliminary data to test whether PTEN is required in adult HSCs to maintain an unusually low level of protein synthesis and whether increased protein synthesis after PTEN deletion induces the tumor suppressor response that depletes HSCs. This work has the potential to yield new techniques to study protein synthesis in rare cell populations in vivo and to open new areas of inquiry related to the role of regulated protein synthesis in hematopoiesis and stem cell function. Defects in the regulation of protein synthesis could potentially contribute to diverse and poorly understood diseases of the hematopoietic system.